The present invention is directed to novel inhibitors of cysteine or serine proteases, referred to herein as hydroxamates. The present invention is also directed to methods for making these novel compounds, and methods for using the same.
Numerous cysteine and serine proteases have been identified in human tissues. A xe2x80x9cproteasexe2x80x9d is an enzyme which degrades proteins into smaller components (peptides). The terms xe2x80x9ccysteine proteasexe2x80x9d and xe2x80x9cserine proteasexe2x80x9d refer to proteases which are distinguished by the presence therein of a cysteine or serine residue which plays a critical role in the catalytic process. Mammalian systems, including humans, normally degrade and process proteins via a variety of enzymes including cysteine and serine proteases. However, when present at elevated levels or when abnormally activated, cysteine and serine proteases may be involved in pathophysiological processes.
For example, calcium-activated neutral proteases (xe2x80x9ccalpainsxe2x80x9d) comprise a family of intracellular cysteine proteases which are ubiquitously expressed in mammalian tissues. Two major calpains have been identified; calpain I and calpain II. While calpain II is the predominant form in many tissues, calpain I is thought to be the predominant form in pathological conditions of nerve tissues. The calpain family of cysteine proteases has been implicated in many diseases and disorders, including neurodegeneration, stroke, Alzheimer""s, amyotrophy, motor neuron damage, acute central nervous system injury, muscular dystrophy, bone resorption, platelet aggregation, cataracts and inflammation. Calpain I has been implicated in excitatory amino-acid induced neurotoxicity disorders including ischemia, hypoglycemia, Huntington""s Disease, and epilepsy. The lysosomal cysteine protease cathepsin B has been implicated in the following disorders: arthritis, inflammation, myocardial infarction, tumor metastasis, and muscular dystrophy. Other lysosomal cysteine proteases include cathepsins C, H, L and S. Interleukin-1xcex2 converting enzyme (xe2x80x9cICExe2x80x9d) is a cysteine protease which catalyzes the formation of interleukin-1xcex2. Interleukin-1xcex2 is an immunoregulatory protein implicated in the following disorders: inflammation, diabetes, septic shock, rheumatoid arthritis, and Alzheimer""s disease. ICE has also been linked to apoptotic cell death of neurons, which is implicated in a variety of neurodegenerative disorders including Parkinson""s disease, ischemia, and amyotrophic lateral sclerosis (ALS).
Cysteine proteases are also produced by various pathogens. The cysteine protease clostripain is produced by Clostridium histolyticum. Other proteases are produced by Trpanosoma cruzi, malaria parasites Plasmodium falciparum and P.vinckei and Streptocococcus. Hepatitis A viral protease HAV C3 is a cysteine protease essential for processing of picornavirus structural proteins and enzymes.
Exemplary serine proteases implicated in degenerative disorders include thrombin, human leukocyte elastase, pancreatic elastase, chymase and cathepsin G. Specifically, thrombin is produced in the blood coagulation cascade, cleaves fibrinogen to form fibrin and activates Factor VIII; thrombin is implicated in thrombophlebitis, thrombosis and asthma. Human leukocyte elastase is implicated in tissue degenerative disorders such as rheumatoid arthritis, osteoarthritis, atherosclerosis, bronchitis, cystic fibrosis, and emphysema. Pancreatic elastase is implicated in pancreatitis. Chymase, an enzyme important in angiotensin synthesis, is implicated in hypertension, myocardial infarction, and coronary heart disease. Cathepsin G is implicated in abnormal connective tissue degradation, particularly in the lung.
Hydroxamates which are structurally distinct from the compounds disclosed herein have been described as inhibitors of glycogen phosphorylase (International Patent Application Pub. No. WO 96/39385) and thrombin (U.S. Pat. No. 5,563,127).
Given the link between cysteine and serine proteases and various debilitating disorders, compounds which inhibit these proteases would be useful and would provide an advance in both research and clinical medicine. The present invention is directed to these, as well as other, important ends.
The present invention is directed to novel cysteine and serine protease inhibitors referred to herein as hydroxamates. In preferred embodiments, the novel compounds are represented by the following Formula I: 
wherein:
W is Axe2x80x94Bxe2x80x94D;
A is aryl(CH2)n, heteroaryl(CH2)n, alkyl having from one to about 14 carbons, alkenyl having from two to about 14 carbons, cycloalkyl having from 3 to about 10 carbons, said A group being optionally substituted with one or more J groups;
B is a bond or CO, SO, SO2, OCO, NR5CO, NR5SO2, or NR5SO;
D is a bond, an amino acid residue, or a peptide composed of 2 to about 5 amino acid residues, said amino acid residue(s) being independently defined by the formula xe2x80x94NHxe2x80x94**CH(R6)xe2x80x94COxe2x80x94, in which ** denotes the xcex1 carbon of an xcex1-amino acid residue possessing, when R6 is other than hydrogen, the D-configuration, the L-configuration, or a mixture of D- and L-;
n is an integer from 0 to about 6;
R1, R2, R3, R4, R5 and R6 are, independently, hydrogen, alkyl having from one to about 14 carbons, cycloalkyl having from 3 to about 10 carbons, said alkyl, and cycloalkyl groups being optionally substituted with one or more J groups; and
J is halogen, lower alkyl, aryl, heteroaryl, haloaryl, amino optionally substituted with one to three aryl or lower alkyl groups, guanidino, alkoxycarbonyl, amido, lower alkylamido, sulfonamido, lower alkyl sulfonamido, lower alkylsulfonyl, lower alkylsulfoxy, lower alkylthio, lower alkoxy, aryloxy, arylalkyloxy, hydroxy, carboxy, cyano, or nitro; and
* denotes the xcex1 carbon of an xcex1-amino acid residue possessing, when R2 is other than hydrogen, the D-configuration, the L-configuration, or a mixture of the D- and L-configurations.
In some preferred embodiments, R1 is alkyl or alkyl substituted with J, wherein J is lower alkoxy. In more preferred embodiments, R1 is benzyl, methoxymethyl, or butyl.
In further preferred embodiments, R2 is alkyl or alkyl substituted with J wherein J is arylalkyloxy or aryl. In more preferred embodiments, R2 is isobutyl or benzyloxymethyl.
In further preferred embodiments, R3 is H.
In some preferred embodiments, R4 is alkyl, alkyl substituted with J, cycloalkyl, or cycloalkyl substituted with J wherein J is aryl, haloaryl, alkyl or heteroaryl. More preferably, R4 is methyl, ethyl, propyl, butyl, benzyl, (pentafluorophenyl)methyl, tert-butyl, or 4-methylcyclohexyl.
In some preferred embodiments, W is benzyloxycarbonyl, methanesulfonyl, benzoyl, tert-butoxycarbonyl, or benzyloxycarbonyl-leucyl.
In some preferred embodiments, R3 is H, and R1 is alkyl or alkyl substituted with J, wherein J is lower alkoxy.
In further preferred embodiments, R3 is H, and R2 is alkyl or alkyl substituted with J wherein J is arylalkyloxy or aryl.
In still further preferred embodiments, R3 is H, and R4 is alkyl, alkyl substituted with J, cycloalkyl, or cycloalkyl substituted with J wherein J is aryl, alkyl, haloaryl, or heteroaryl.
In still further preferred embodiments, R3 is H, R1 is alkyl or alkyl substituted with J, wherein J is lower alkoxy, and R2 is alkyl or alkyl substituted with J wherein J is arylalkyloxy or aryl.
In still further preferred embodiments, R3 is H, R1 is alkyl or alkyl substituted with J, wherein J is lower alkoxy, and R4 is alkyl, alkyl substituted with J, cycloalkyl, or cycloalkyl substituted with J wherein J is aryl, haloaryl, alkyl or heteroaryl.
In further preferred embodiments, R3 is H, R1 is alkyl or alkyl substituted with J, wherein J is lower alkoxy, R4 is alkyl, alkyl substituted with J, cycloalkyl, or cycloalkyl substituted with J wherein J is aryl, haloaryl, alkyl or heteroaryl, and R2 is alkyl or alkyl substituted with J wherein J is arylalkyloxy or aryl.
In some particularly preferred embodiments, R1 is benzyl, methoxymethyl, or butyl; R2 is isobutyl or benzyloxymethyl; R3 is hydrogen; R4 is methyl, ethyl, propyl, butyl, benzyl, (pentafluorophenyl)methyl, tert-butyl, or 4-methylcyclohexyl; and W is benzyloxycarbonyl, methanesulfonyl, benzoyl, tert-butoxycarbonyl, or benzyloxycarbonyl-leucyl.
In further particularly preferred embodiments, R1 is benzyl; R2 is isobutyl; * denotes the xcex1 carbon of an xcex1-amino acid residue possessing the L-configuration; R3 is hydrogen; R4 is methyl, ethyl, propyl, butyl, benzyl, (pentafluorophenyl)methyl, tert-butyl, or 4-methylcyclohexyl; and W is benzyloxycarbonyl or benzyloxycarbonyl-leucyl.
In further particularly preferred embodiments, R1 is benzyl; R2 is benzyloxymethyl; * denotes the xcex1 carbon of an xcex1-amino acid residue possessing the D-configuration; R3 is hydrogen; R4 is methyl, ethyl, or benzyl; and W is methanesulfonyl.
Some especially preferred embodiments of the invention are described in Table 1, infra.
The present invention also provides compositions for inhibiting a protease selected from the group consisting of serine proteases and cysteine proteases comprising a compound of the invention.
Also provided by the present invention are methods for inhibiting a protease comprising contacting a protease selected from the group consisting of serine proteases and cysteine proteases with an inhibitory amount of a compound of the invention, and methods for inhibiting a protease comprising contacting a protease selected from the group consisting of serine proteases and cysteine proteases with an inhibitory amount of a composition comprising a compound of the invention.
The compounds of the invention are useful for inhibition of cysteine and serine proteases. Beneficially, these compounds find utility in a variety of settings. For example, in the research arena, the claimed compounds can be used, for example, in discovery of agents for treating disorders associated with abnormal and/or aberrant activity of cysteine and/or serine proteases. In a clinical arena, for example, the compounds can be used to alleviate, mediate, reduce, and/or prevent disorders which are associated with abnormal and/or aberrant activity of cysteine and/or serine proteases.
Thus, in some preferred embodiments, the present invention further provides pharmaceutical compositions comprising a compound of the invention, preferably also containing a pharmaceutically acceptable carrier. Also provided in accordance with the present invention are compositions for the treatment of a disorder, which is preferably neurodegeneration, stroke, Alzheimer""s, amyotrophy, motor neuron damage, acute central nervous system injury, muscular dystrophy, bone resorption, platelet aggregation, cataracts and inflammation, comprising a compound of claim 1 and a pharmaceutically effective carrier. The present invention also provides methods for the treatment of a disorder, which is preferably neurodegeneration, stroke, Alzheimer""s, amyotrophy, motor neuron damage, acute central nervous system injury, muscular dystrophy, bone resorption, platelet aggregation, cataracts and inflammation, comprising administering to a subject in need of such treatment an effective amount of a compound of the invention
Because the hydroxamates of the invention inhibit cysteine proteases and serine proteases, they can be used in both research and therapeutic settings. These and other features of the compounds of the subject invention are set forth in more detail below.
The present invention provides novel inhibitors of cysteine and serine protease inhibitors. In preferred embodiments, the compounds of the invention have the Formula I: 
wherein:
W is Axe2x80x94Bxe2x80x94D;
A is aryl(CH2)n, heteroaryl(CH2)n, alkyl having from one to about 14 carbons, alkenyl having from two to about 14 carbons, cycloalkyl having from 3 to about 10 carbons, said A group being optionally substituted with one or more J groups;
B is a bond or CO, SO, SO2, OCO, NR5CO, NR5SO2, or NR5SO;
D is a bond, an amino acid residue, or a peptide composed of 2 to about 5 amino acid residues, said amino acid residue(s) being independently defined by the formula xe2x80x94NHxe2x80x94**CH(R6)xe2x80x94COxe2x80x94, in which ** denotes the xcex1 carbon of an xcex1-amino acid residue possessing, when R6 is other than hydrogen, the D-configuration, the L-configuration, or a mixture of D- and L-;
n is an integer from 0 to about 6;
R1, R2, R3, R4, R5 and R6 are, independently, hydrogen, alkyl having from one to about 14 carbons, cycloalkyl having from 3 to about 10 carbons, said alkyl, and cycloalkyl groups being optionally substituted with one or more J groups; and
J is halogen, lower alkyl, aryl, heteroaryl, haloaryl, amino optionally substituted with one to three aryl or lower alkyl groups, guanidino, alkoxycarbonyl, amido, lower alkylamido, sulfonamido, lower alkyl sulfonamido, lower alkylsulfonyl, lower alkylsulfoxy, lower alkylthio, lower alkoxy, aryloxy, arylalkyloxy, hydroxy, carboxy, cyano, or nitro; and
* denotes the xcex1 carbon of an xcex1-amino acid residue possessing, when R2 is other than hydrogen, the D-configuration, the L-configuration, or a mixture of the D- and L-configurations.
The compounds of the invention are useful in a variety of settings. For example, in a research environment, preferred compounds having defined attributes can be used to screen for natural and synthetic compounds which evidence similar characteristics in inhibiting protease activity. Inhibition of cysteine protease or serine protease activity can be measured by determining the rate of inactivation of a protease using a compound of the invention. The compounds can also be used in the refinement of in vitro and in vivo models for determining the effects of inhibition of particular proteases on particular cell types or biological conditions. In a therapeutic setting, given the connection between cysteine proteases and certain defined disorders, and serine proteases and certain defined disorders, compounds of the invention can be utilized to alleviate, mediate, reduce and/or prevent disorders which are associated with abnormal and/or aberrant activity of cysteine proteases and/or serine proteases.
As used herein, the term xe2x80x9calkylxe2x80x9d is meant to include straight-chain, branched and cyclic hydrocarbon groups such as, for example, ethyl, and isopropyl groups. Preferred alkyl groups have 1 to about 10 carbon atoms. The term xe2x80x9clower alkylxe2x80x9d refers to alkyl groups of 1-6 carbon atoms. In general, the term xe2x80x9clowerxe2x80x9d refers to groups having up to six carbon atoms. The term xe2x80x9ccycloalkylxe2x80x9d denotes cyclic alkyl groups, such as, for example, cyclopropyl groups. The term xe2x80x9calkenylxe2x80x9d denotes alkyl groups that contain at least one double bond. xe2x80x9cArylxe2x80x9d groups are aromatic cyclic compounds including but not limited to phenyl, tolyl, naphthyl, anthracyl, phenanthryl, pyrenyl, and xylyl. Preferred aryl groups include phenyl and naphthyl.
In general, the term xe2x80x9cheteroxe2x80x9d when used as a prefix denotes the presence of one or more hetero atoms such as O, N or S. Thus, the term xe2x80x9cheterocyclicxe2x80x9d refers to cyclic groups in which the ring portion includes at least one heteroatom. xe2x80x9cHeteroalkylxe2x80x9d groups are heterocycles containing solely single bonds within their ring portions, i.e. saturated heteroatomic ring systems. The term xe2x80x9cheteroarylxe2x80x9d denotes aryl groups wherein at least one ring carbon has been replaced with a hetero atom. The term xe2x80x9chaloarylxe2x80x9d is intended to mean an aryl group that bears one or more halogen atoms.The term xe2x80x9chalogenxe2x80x9d refers to F, Cl, Br, and I atoms.
As used herein, xe2x80x9calkoxyxe2x80x9d groups are alkyl groups linked through an oxygen atom. Examples of alkoxy groups include methoxy (xe2x80x94OCH3) and ethoxy (xe2x80x94OCH2CH3) groups. In general, the term xe2x80x9coxyxe2x80x9d when used as a suffix denotes attachment through an oxygen atom. Thus, alkoxycarbonyl groups are carbonyl groups which contain an alkoxy substituent, i.e., groups of general formula xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94R, where R is alkyl. The term xe2x80x9caryloxyxe2x80x9d denotes an aryl group linked through an oxygen atom. The term xe2x80x9carylalkylxe2x80x9d (or xe2x80x9caralkylxe2x80x9d) denotes an alkyl group that bears an aryl substituent. The term xe2x80x9carylalkyloxy (or xe2x80x9caralkyloxyxe2x80x9d) denotes an aralkyl group linked through an oxygen atom.
As used herein, the term xe2x80x9camino acidxe2x80x9d denotes a molecule or residue thereof containing both an amino group and xcex1 carboxyl group. As used herein the term xe2x80x9cxcex1-amino acidxe2x80x9d means an amino acid of general formula HOOCxe2x80x94CH(sidechain)-NH2, or a residue of such amino acid of formula, for example, xe2x80x94C(xe2x95x90O)xe2x80x94CH(sidechain)xe2x80x94NHxe2x80x94. In preferred embodiments of the compounds of the invention, the xcex1-carbon (i.e., the carbon that bears the sidechain) of constituent amino acids can be exclusively in the L-configuration, exclusively in the D-configuration, or in a mixture of D and L configurations in any proportion.
Functional groups present on the compounds of Formula I may contain protecting groups. For example, the amino acid sidechain substituents of the compounds of Formula I can be substituted with protecting groups such as benzyloxycarbonyl or t-butoxycarbonyl groups. Protecting groups are known per se as chemical functional groups that can be selectively appended to and removed from functionalities, such as hydroxyl groups and carboxyl groups. These groups are present in a chemical compound to render such functionality inert to chemical reaction conditions to which the compound is exposed. Any of a variety of protecting groups may be employed with the present invention. One such protecting group is the benzyloxycarbonyl (Cbz; Z) group. Other preferred protecting groups according to the invention may be found in Greene, T. W. and Wuts, P. G. M., xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d 2d. Ed., Wiley and Sons, 1991.
As used herein, the term xe2x80x9camidoxe2x80x9d has its accustomed meaning as a group of formula xe2x80x94C(xe2x95x90O)xe2x80x94NHxe2x80x94. The term xe2x80x9calkylamidoxe2x80x9d denotes an amido grouop that bears an alkyl substituent. The term xe2x80x9csulfonamidoxe2x80x9d denotes a group of formula xe2x80x94SO2xe2x80x94NHxe2x80x94. In general, the term xe2x80x9calkylxe2x80x9d or xe2x80x9carylxe2x80x9d when used as a prefix in such terms as xe2x80x9calkylsulfonamido,xe2x80x9d xe2x80x9calkylsulfonyl,xe2x80x9d xe2x80x9calkylsulfoxyxe2x80x9d or xe2x80x9calkylthioxe2x80x9d indicates that the sulfonamido, sulfonyl, sulfoxy or thio group bears an alkyl substituent.
Some constituent groups represented in the Formulas described herein can be substituted. As used herein, the term xe2x80x9csubstitutedxe2x80x9d indicates that any available hydrogen atom of the moiety designated as xe2x80x9csubstitutedxe2x80x9d can be replaced by the indicated group.
In preferred embodiments, compositions are provided for inhibiting a serine protease or a cysteine protease comprising a compound of the invention. In other preferred embodiments, methods are provided for inhibiting serine proteases or cysteine proteases comprising contacting a protease selected from the group consisting of serine proteases and cysteine proteases with an inhibitory amount of a compound of the invention.
The disclosed compounds of the invention are useful for the inhibition of cysteine proteases and serine proteases. As used herein, the terms xe2x80x9cinhibitxe2x80x9d and xe2x80x9cinhibitionxe2x80x9d mean having an adverse effect on enzymatic activity. An inhibitory amount is an amount of a compound of the invention effective to inhibit a cysteine and/or serine protease.
Pharmaceutically acceptable salts of the cysteine and serine protease inhibitors also fall within the scope of the compounds as disclosed herein. The term xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d as used herein means an inorganic acid addition salt such as hydrochloride, sulfate, and phosphate, or an organic acid addition salt such as acetate, maleate, fumarate, tartrate, and citrate. Examples of pharmaceutically acceptable metal salts are alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt, and zinc salt. Examples of pharmaceutically acceptable ammonium salts are ammonium salt and tetramethylammonium salt. Examples of pharmaceutically acceptable organic amine addition salts are salts with morpholine and piperidine. Examples of pharmaceutically acceptable amino acid addition salts are salts with lysine, glycine, and phenylalanine.
Compounds provided herein can be formulated into pharmaceutical compositions by admixture with pharmaceutically acceptable nontoxic excipients and carriers. As noted above, such compositions may be prepared for use in parenteral administration, particularly in the form of liquid solutions or suspensions; or oral administration, particularly in the form of tablets or capsules; or intranasally, particularly in the form of powders, nasal drops, or aerosols; or dermally, via, for example, transdermal patches; or prepared in other suitable fashions for these and other forms of administration as will be apparent to those skilled in the art.
The composition may conveniently be administered in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art, for example, as described in Remington""s Pharmaceutical Sciences (Mack Pub. Co., Easton, Pa., 1980). Formulations for parenteral administration may contain as common excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils and vegetable origin, hydrogenated naphthalenes and the like. In particular, biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be useful excipients to control the release of the active compounds. Other potentially useful parenteral delivery systems for these active compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation administration contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally. Formulations for parenteral administration may also include glycocholate for buccal administration, a salicylate for rectal administration, or citric acid for vaginal administration. Formulations for transdermal patches are preferably lipophilic emulsions.
The materials of this invention can be employed as the sole active agent in a pharmaceutical or can be used in combination with other active ingredients, e.g., other growth factors which could facilitate neuronal survival or axonal regeneration in diseases or disorders.
The concentrations of the compounds described herein in a therapeutic composition will vary depending upon a number of factors, including the dosage of the drug to be administered, the chemical characteristics (e.g., hydrophobicity) of the compounds employed, and the route of administration. In general terms, the compounds of this invention may be provided in effective inhibitory amounts in an aqueous physiological buffer solution containing about 0.1 to 10% w/v compound for parenteral administration. Typical dose ranges are from about 1 mg/kg to about 1 g/kg of body weight per day; a preferred dose range is from about 0.01 mg/kg to 100 mg/kg of body weight per day. Such formulations typically provide inhibitory amounts of the compound of the invention. The preferred dosage of drug to be administered is likely, however, to depend on such variables as the type and extent of progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the compound selected, and formulation of the compound excipient, and its route of administration.
As used herein, the term xe2x80x9ccontactingxe2x80x9d means directly or indirectly causing at least two moieties to come into physical association with each other. Contacting thus includes physical acts such as placing the moieties together in a container, or administering moieties to a patient. Thus, for example administering a compound of the invention to a human patient evidencing a disease or disorder associated with abnormal and/or aberrant activity of such proteases falls within the scope of the definition of the term xe2x80x9ccontactingxe2x80x9d.
The invention is further illustrated by way of the following examples which are intended to elucidate the invention. These examples are not intended, nor are they to be construed, as limiting the scope of the disclosure.